Facial symmetry is a universal measure of human facial attractiveness. This trait evolves under intense sexual selection as a signal of robust physical health and genetic quality in potential mates. In spite of this, deviations from perfect facial symmetry are extremely common in the human population. Craniofacial (CF) asymmetries manifest themselves in both soft- and hard-tissues of the head, and demonstrate a spectrum of severity from subclinical to severe. Abnormal asymmetries can cause impaired dental function (e.g., cross bite), altered esthetic appearance, ankylosis of the temporomandibular joint, and breathing impairments threatening survival of affected children. Clinical diagnosis and treatments are advanced, however very little is known of the genetic, molecular and developmental aberrations that underlie CF asymmetries. Highly invasive surgical intervention and reconstruction are often required to correct asymmetries at delicate life stages. Therefore, an improved understanding of the regulators of abnormal asymmetry will enable earlier therapeutic corrective options and identify risk loci for prenatal diagnosis. Abnormal asymmetries are difficult to study in humans or traditional model systems owing to their unpredictability, random appearance along the left-right (L/R) axis, and variable penetrance. We will utilize a powerful natural system (Astyanax mexicanus) consisting of conspecific cave- and surface-dwelling fish separated by >1My of evolution. Like humans, cavefish harbor random and variably penetrant CF asymmetries under genetic control. Despite their randomness, certain forms of CF asymmetry are exceedingly common in cavefish, rendering them a powerful tool for investigation. This unique natural experiment enables direct comparison between asymmetric (cave) morphs and perfectly symmetric (surface) morphs to uncover the precise genetic, molecular and developmental differences causing and accompanying asymmetry. We hypothesize breakage of L/R symmetry reflects departures from normal symmetric gene expression across the L/R axis. We anticipate that L/R alterations in cranial neural crest cell development, specifically at the differentiation stage of osteogenic precursors, prefigure L/R asymmetries in derived cranial tissues. Finally, we predict that unilateral manipulation of bmp4 and/or tgfb3 signaling will recapitulate asymmetric abnormalities in a normally symmetric organism. The PI has an established record of quantitative genetic analyses using this system, was awarded preliminary funding through the NIDCR which laid the groundwork for the proposed studies, has significant training in the field of developmental genetics, has a substantial record of productivity/publishing in non-traditional model system research, and is a collaborator on the Astyanax Genome Sequencing Project. Each of these components will help ensure the success of the proposed studies. Future work will connect transcriptomic variation to tissue level alterations (apoptosis, progenitor specification), determine how genetic changes impact cellular/developmental asymmetries, and inform how genetic changes influence transcriptional effects causing L/R asymmetries.